Biopharmaceutical manufacturers continue to seek new efficiencies through the development of higher-titer fed-batch production processes, which require innovations in media combinations, feeding options, and screening strategies to meet the high and complex nutrient demands for growth in fed-batch operations. Jonathan Kaiser, Senior Scientist in Innovation Cell Culture Media Development and BioProcessing R&D at MilliporeSigma, discusses how manufacturers can optimize fed-batch production and the advantages of MilliporeSigma’s media and feed systems, with Pharma’s Almanac Editor in Chief David Alvaro, Ph.D.
David Alvaro (DA): To start off, can you give a brief overview of fed-batch processes and their unique advantages?
Jonathan Kaiser (JK): Fed-batch processes are ultimately more finite and straightforward than continuous and perfusion processes. After a fed-batch experiment is seeded, it quickly enters into an exponential growth phase, which is followed later by a stationary phase, and the whole process takes place over a relatively short period of about 14 days. The production medium is used to initiate the culture and provide for the initial growth, and then a feed is added to replenish nutrients as they’re consumed, sustaining the culture and driving production.
A primary differentiator of fed-batch approaches is that only additions are made to the culture vessel and recovery occurs only at the very end, in contrast to continuous or perfusion processes, which add a single medium on a continuous basis throughout the process. As media is added, it is also removed, which allows you to remove product and by-product — desired and undesired — on a regular basis. While there are multiple perfusion approaches, they all rely on having a separate cell retention device within the process. As a result, they are generally more complex to execute and often take longer to complete.
Fed-batch is the basis of many legacy processes and continues to be leveraged in new process development, in large part because of that reduced complexity. It can generate product in a relatively short period of time. It doesn’t require retention devices or extra pumps and uses a relatively low amount of media compared with continuous processes.
Perfusion has real advantages under conditions where the residence time of the product in the vessel is important. For instance, if a product has low stability or is susceptible to modification, perfusion is useful, because of that continuous removal of product, which can then be processed quickly.
DA: What novel challenges do fed-batch processes present?
JK: One of the big challenges for fed-batch involves delivering the right mix of raw materials at the right time to both sustain the culture and drive production. A medium and feed needs to work in combination to ensure that essential nutrients are not completely exhausted over the course of the run — a run that typically includes multiple stages, and where we don’t get to remove anything during the process. Customers would prefer a single feed solution to serve their entire run rather than a multipart feed solution. Since we can’t take anything out of the process, we need to make sure that toxic by-products, such as ammonia, don’t accumulate and that the osmolality does not increase to the point of toxicity. We also need to monitor glucose to prevent exhaustion, which will quickly take any process down. Some customers prefer to have very tight control over the glucose in their process.
DA: In a broad sense, what options are available to address those challenges?
JK: These challenges have led to the creation of chemically defined media and feeds with consistent composition, which allows for tight control of nutrients delivered to the medium and subsequently predictable delivery of additional nutrients through the feed. At MilliporeSigma, we have full knowledge and control over the composition of all of our products.
To accommodate the diversity of CHO cell systems and clones, the timing of feed administration and the amount of feed administration are often varied. Different manufacturers have different recommendations tied to their particular products, but individual customers also have preferences in terms of how they approach percentage and delivery over time. In addition, the growth profile of clones varies in speed, amplitude, and longevity. Even in very consistent development processes, CHO systems produce a variety of clones with different characteristics. However, even those characteristics, such as growth, are not universal predictors of cellular preferences in terms of things like media and feed. Multiple media and feed solutions have been developed, along with screening methods to address this level of diversity.
DA: Even though fed-batch has been around for some time, do approaches continue to evolve and demand new media and feed solutions?
JK: Even after all of these years of fed-batch, products continue to evolve to support the ongoing evolution of different fed-batch approaches. Traditionally, fed-batch cultures began with a relatively low initial viable cell density and were allowed to grow from there. Within the industry, there’s a push toward intensified processes that use higher initial viable cell densities while trying to shorten run length. Higher cell density leads to more productivity, and shorter runs increase the total number of runs that can be performed in a given period of time, leading to more product overall. Suppliers like MilliporeSigma realized the need to develop products that support these kinds of approaches through concentrated feeds, which have become the standard for catalogue products, since lower-concentrated feeds are challenged to deliver enough nutrients to deal with the higher cell densities.
At the same time, there’s a push within the industry to simplify processes as much as possible. Feeding a fed-batch can be accomplished either through the more traditional approach of a two-part feed system or the more modern approach of a one-part system. In a two-part system, a neutral pH feed delivers the majority of nutrients and is combined with a high pH feed that delivers nutrients that aren’t especially soluble at the neutral pH — the two feeds need to be hydrated, stored, and fed separately. In a one-part system, such as Cellvento® ModiFeed Prime COMP, all the nutrients required are delivered through a single feed.
DA: How do manufacturer evaluate feeds to determine which is best suited to a particular process?
JK: There is no single solution in terms of medium and feed; no single approach that will be optimal for all CHO cells. Customers often evaluate different solutions from a variety of different vendors, testing various platforms alone or mixing and matching media and feeds from different vendors — sometimes even two or more media or feeds from different vendors are mixed into a single solution. However, a mixture of products from different vendors can become a serious sourcing challenge in later stages of development. At MilliporeSigma, we recommend that customers consider the long-term implications of what is often only small-scale evaluation of different platforms.
In addition, it’s really important when evaluating platforms to evaluate a variety of different clones that are representative of your system or goals. There can always be an outlier in cellular preferences, especially with CHO, so you really need a variety of representative clones. Ideally, if time allows, it’s best to adapt your cells to the medium that you’re trying to evaluate, because that will be the best representation of future performance and will assure that your process will translate well into the future.
We also recommend that customers evaluate multiple media options in the selection process, because different pairings of media and clones may react differently to feeds. Screening multiple feeds at different total percentages and using different schedules can capture the variety of cellular preferences tied to nutrient levels. The ultimate assessment of performance for a medium and feed pair relies on outcomes like productivity, longevity, viable cell density, and, in many cases, product quality. At MilliporeSigma, we offer a variety of solutions for this kind of evaluation that allow customers to work with a single supplier and feel more confident about how the evaluation results will determine outcomes at later stages.
MilliporeSigma has a group of technologically knowledgeable individuals within our organization called Manufacturing Sciences and Technology (MSAT) that is designed to be more closely associated with the customer, to support their evaluation of our products and help them utilize them in the most efficient manner possible.
DA: Can you expand on the solutions MilliporeSigma offers with your EX-CELL® and Cellvento® product lines?
JK: The EX-CELL® Advanced and Cellvento® platforms are MilliporeSigma’s flagship fed-batch platforms for suspension CHO. The EX-CELL® Advanced platform is a fed-batch medium and feed pair that offers a diverse set of raw materials and is integrated into our CHOZN® expression platform to serve as the production medium and feed. EX-CELL® Advanced CHO Feed 1 is formulated at a relatively low final grams per liter concentration and fed at a relatively high total feed percentage. The Cellvento® platform is a fed-batch medium and feed pair containing a highly concentrated one-part feed.
To these existing platforms, we recently introduced Cellvento® ModiFeed Prime COMP, which is a new, highly concentrated one-part feed compatible with both flagship media for CHO. Cellvento® 4Feed COMP and Cellvento® ModiFeed Prime COMP are both highly concentrated feeds. All three feeds are available in a glucose-free format, because we want to give customers as much control over this parameter as they would like.
The two platforms can be used interchangeably and in combination to serve the diverse range of CHO cell preferences and characteristics. If a particular clone needs a greater supply of raw materials, it may be best served by Cellvento® 4Feed COMP, but if it instead requires more diverse supply of raw materials, it may be best suited to EX-CELL® Advanced CHO Feed 1. As a single solution, Cellvento® ModiFeed Prime COMP brings together the best of these characteristics.
EX-CELL® Advanced CHO Feed 1 and Cellvento® 4Feed COMP can even be mixed in a variety of ratios, such as 25:75, 50:50 or 75:25, for optimization across the diversity of CHO cells. Attention should be paid to all outputs of interest, as each medium and feed can affect output beyond productivity, including attributes like protein quality. For example, EX-CELL® Advanced CHO medium tends to produce products with more higher order glycans, while Cellvento® 4CHO COMP tends to yield products with fewer mature glycans, which can be important attributes in many cases.
One additional simplification aspect that merits mention is the desire, on the part of customers, for increased flexibility in how their feeds are utilized. Customers want to put their focus on the production process once it is underway. To facilitate this, a fed-batch feed would ideally be flexible enough that the customer could hydrate it, leave it out at room temperature, connect it to a bioreactor, and just feed as needed on demand. This eliminates the need to risk contamination by making multiple connections over the course of the run or needing to go back and forth between cold storage. At MilliporeSigma, we are striving to facilitate room temperature applicability of our products as much as possible. Hydrated Cellvento® ModiFeed Prime COMP, if stored protected from light, can be kept at room temperature for up to 30 days; unlike most feeds, which require 2–8 °C storage. Few other suppliers choose to be forward in a statement of their product’s ability to withstand room temperature storage.
DA: To what degree are these media and feed systems optimized for MilliporeSigma’s CHOZN® platform, and how applicable are they to other CHO systems?
JK: The EX-CELL® Advanced platform is currently recommended for the fed-batch confirmation of clones developed through the CHOZN® platform. The EX-CELL® Advanced CHO medium was specifically engineered to transition CHOZN® clones from the selection phase, which uses EX-CELL® CD CHO Fusion media, into production. Following the selection of final CHOZN® clones, any of MilliporeSigma’s modern fed-batch solutions can be utilized. Best results are often obtained by keeping the CHOZN® clones in EX-CELL® Advanced CHO medium, but some clones coming out of the CHOZN® expression system can adapt into Cellvento® 4CHO COMP, which creates additional opportunities for customers. Cellvento® ModiFeed Prime COMP, which was developed using both platforms and yields superior performance, offers an additional option for high-performing CHOZN® clones.
The CHOZN® expression system is an important piece of the overall CHO marketplace, but all of our fed-batch platforms extend beyond just one system. We develop our products using cell lines from a variety of lineages, such as CHO-K1, DG44, and CHO-S. MilliporeSigma encourages customers to experience the advantages of our fed-batch platforms, no matter what CHO system they use.
DA: After determining the optimal media and feed for your cells, what additional optimization work should a manufacturer perform to get the most out of the system?
JK: You can pretty quickly select the ideal medium and feed system to match the preference of your clone, but we recommend that customers evaluate as many conditions as possible to maximize success in terms of not only the right combination of medium and feed but the right feed schedule and right total feed percentage. With Cellvento® ModiFeed Prime COMP in particular, we recommend evaluating both a high and a low total feed percentage to capture cell preferences for more or less total feed, since you can’t always predict what will be preferred by your cell line. Once this kind of evaluation gives the customer an idea of what their cell line prefers, they can even go higher or lower to determine the level for optimal productivity. Combining EX-CELL® Advanced CHO Feed 1 and Cellvento® 4Feed COMP in a variety of ratios makes it easy to capture a range of diversity in raw material preferences. We offer customers a variety of literature to help them understand these evaluations and mixing possibilities.
DA: Can you share anything about how you see the available design space for further media feed products and the ongoing R&D work at MilliporeSigma?
JK: MilliporeSigma is always working on the next innovation in cell culture media, specifically in the area of fed-batch. We currently have a program seeking to further concentrate fed-batch feeds. Cellvento® 4Feed COMP and Cellvento® ModiFeed Prime COMP are in the 130 g/L range, excluding glucose. Going even further in terms of concentration should allow seeding of cultures at even higher volumes, because they will require less volume for the feed, and this should ultimately lead to higher productivity over the same run period.
In addition, we’re working on solutions to further decrease the variability of some of the raw materials that are possible sources of trace elements. Even within the world of chemically defined media and feeds, we strive to eliminate as much variability as possible through consistent raw materials. Consistent composition contributes to highly consistent processes and consistent final product.
Finally, we’re developing solutions to allow customers to easily regulate their glycosylation profiles. Whether a customer is developing an innovator molecule or a biosimilar, protein quality is critical, and the correct glycosylation pattern can increase the efficacy of innovator molecules or make biosimilar development possible by matching the profile of the originator molecule. MilliporeSigma wants to be the provider of choice for glycosylation solutions. We are looking to further concentrate feeds, increase the consistency of raw materials, and support customers in their desire for particular glycosylation profiles.
DA: Is there a concise take-home message you’d like to leave readers with?
JK: MilliporeSigma strives to deliver novel products and approaches to address prevailing industry challenges, namely: speeding up development, increasing process efficiency, and reducing complexity. We also facilitate process optimization, risk assessment, and quality efforts through our EMPROVE® Program. We are an experienced industry partner with a holistic view on the entire biopharmaceutical process.
Recommended reading
- Application Note: Optimizing Feed Strategies with Cellvento® ModiFeed Prime COMP: A Scalable Process. 2022.
- Kaiser, Jonathan and Brandl, Melanie. “Improving fed-batch yields by combining EX-CELL® Advanced and Cellvento® cell culture media portfolios.” MilliporeSigma. 2022.